This invention is in the field of plant molecular biology. More specifically, this invention pertains to nucleic acid fragments encoding stearoyl desaturases in plants and seeds.
Stearoyl-acyl carrier protein (stearoyl-ACP) desaturase, also known as delta-9 desaturase (EC 1.14.99.6), catalyzes the first step in C-18 fatty acid desaturation in plants. This reaction converts stearic acid to oleic acid (stearoyl-ACP to oleoyl-ACP) by introducing a double bond at the 9/10 position of the C18 chain. cDNAs encoding stearoyl-ACP desaturase have been isolated from several plants including safflower, castor, rapeseed, canola, rice and soybean. Different isozymes may be useful for changing the sturated fatty acid content in seed oil.
The oil of prickly poppy is rich in 9-oxooctacosanoate, a delta-9 keto fatty acid. A prickly poppy gene with similarity to stearoyl-ACP desaturase may encode an enzyme which inserts a keto group rather than a double bond at positon 9, or may provide the substrate for further chemistry to make the keto group. Overexpression of this gene in soybeans, corn, etc. may yield oils rich in keto fatty acids which are useful for industrial applications such as drying agents and monomer precursors.
Stearoyl-CoA desaturase (EC 1.14.99.5) is an integral endoplasmic reticulum membrane protein which introduces a second cis bond at the 12 positon of fatty acids bound to membrane glycerolipids. In rose petals a steroyl-CoA desaturase is induced during senesence. Overexpression of a cytoplasmic acyl-CoA desaturase in seeds may be useful to reduce the levels of saturated CoAs reducing the saturated oils in soybeans and corn.
Overexpression of these desaturases in plants may help increase the cold tolerance since the phase transition temperature of lipids in the cellular membranes depends on the degreee of unsaturation of fatty acids of the membrane lipids.
The present invention relates to isolated polynucleotides comprising a nucleotide sequence encoding a corn stearoyl-ACP desaturase polypeptide of SEQ ID NO:2 and 14, a rice stearoyl-ACP desaturase polypeptide of SEQ ID NO:8 and 20, and a wheat stearoyl-ACP desaturase polypeptide of SEQ ID NO:10 and 22. Additionally, the present invention relates to isolated polynucleotides comprising a nucleotide sequence encoding a first polypeptide of at least 224 amino acids that has at least 86% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of a prickly poppy stearoyl-ACP desaturase polypeptide of SEQ ID NOs:4, 6, 16, and 18. The present invention also relates to isolated polynucleotides comprising a nucleotide sequence encoding a first polypeptide of at least 60 amino acids that has at least 60% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of a tobacco stearoyl-CoA desaturase polypeptide of SEQ ID NOs:12 and 24, and a pot marigold stearoyl-CoA desaturase polypeptide of SEQ ID NO:32 and 34. Further, the present invention relates to isolated polynucleotides comprising a nucleotide sequence encoding a first polypeptide of at least 40 amino acids that has at least 60% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of an Arabidopsis thaliana stearoyl-CoA desaturase polypeptide of SEQ ID NOs:22 and 28, a Peruvian lilly stearoyl-CoA desaturase polypeptide of SEQ ID NO:30, a rice stearoyl-CoA desaturase polypeptide of SEQ ID NO:36, a soybean stearoyl-CoA desaturase polypeptide of SEQ ID NO:38, and a grape stearoyl-CoA desaturase polypeptide of SEQ ID NO:40. The present invention also relates to an isolated polynucleotide comprising the complement of the nucleotide sequences described above.
It is preferred that the isolated polynucleotides of the claimed invention consist of a nucleic acid sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, and 39 that codes for the polypeptide selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, and 40. The present invention also relates to an isolated polynucleotide comprising a nucleotide sequences of at least 40 (preferably at least 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39 and the complement of such nucleotide sequences.
The present invention relates to a chimeric gene comprising an isolated polynucleotide of the present invention operably linked to suitable regulatory sequences.
The present invention relates to an isolated host cell comprising a chimeric gene of the present invention or an isolated polynucleotide of the present invention. The host cell may be eukaryotic, such as a yeast or a plant cell, or prokaryotic, such as a bacterial cell. The present invention also relates to a virus, preferably a baculovirus, comprising an isolated polynucleotide of the present invention or a chimeric gene of the present invention.
The present invention relates to a process for producing an isolated host cell comprising a chimeric gene of the present invention or an isolated polynucleotide of the present invention, the process comprising either transforming or transfecting an isolated compatible host cell with a chimeric gene or isolated polynucleotide of the present invention.
The present invention relates to a stearoyl desaturase polypeptide selected from the group consisting of SEQ ID NOs:2, 8, 10, 14, 20, and 22. Also, the present invention relates to a stearoyl desaturase polypeptide of at least 224 amino acids comprising at least 86% homology based on the Clustal method of alignment compared to a polypeptide selected from the group consisting of SEQ ID NOs:4, 6, 16, and 18. Further, the present invention relates to a stearoyl desaturase polypeptide of at least 60 amino acids comprising at least 60% homology based on the Clustal method of alignment compared to a polypeptide selected from the group consisting of SEQ ID NOs:12, 24, 32, and 34. And still further, the present invention relates to a stearoyl desaturase polypeptide of at least 40 amino acids comprising at least 60% homology based on the Clustal method of alignment compared to a polypeptide selected from the group consisting of SEQ ID NOs:22, 28, 30, 36, 38, and 40.
The present invention relates to a method of selecting an isolated polynucleotide that affects the level of expression of a stearoyl desaturase polypeptide in a host cell, preferably a plant cell, the method comprising the steps of:
constructing an isolated polynucleotide of the present invention or an isolated chimeric gene of the present invention;
introducing the isolated polynucleotide or the isolated chimeric gene into a host cell;
measuring the level a stearoyl desaturase polypeptide in the host cell containing the isolated polynucleotide; and
comparing the level of a stearoyl desaturase polypeptide in the host cell containing the isolated polynucleotide with the level of a stearoyl desaturase polypeptide in a host cell that does not contain the isolated polynucleotide.
The present invention relates to a method of obtaining a nucleic acid fragment encoding a substantial portion of a stearoyl desaturase polypeptide gene, preferably a plant stearoyl desaturase polypeptide gene, comprising the steps of: synthesizing an oligonucleotide primer comprising a nucleotide sequence of at least 60 (preferably at least 40, most preferably at least 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39 and the complement of such nucleotide sequences; and amplifying a nucleic acid fragment (preferably a cDNA inserted in a cloning vector) using the oligonucleotide primer. The amplified nucleic acid fragment preferably will encode a portion of a stearoyl desaturase amino acid sequence.
The present invention also relates to a method of obtaining a nucleic acid fragment encoding all or a substantial portion of the amino acid sequence encoding a stearoyl desaturase polypeptide comprising the steps of: probing a cDNA or genomic library with an isolated polynucleotide of the present invention; identifying a DNA clone that hybridizes with an isolated polynucleotide of the present invention; isolating the identified DNA clone; and sequencing the cDNA or genomic fragment that comprises the isolated DNA clone.
A further embodiment of the instant invention is a method for evaluating at least one compound for its ability to inhibit the activity of a stearoyl desaturase, the method comprising the steps of: (a) transforming a host cell with a chimeric gene comprising a nucleic acid fragment encoding a stearoyl desaturase, operably linked to suitable regulatory sequences; (b) growing the transformed host cell under conditions that are suitable for expression of the chimeric gene wherein expression of the chimeric gene results in production of stearoyl desaturase in the transformed host cell; (c) optionally purifying the stearoyl desaturase expressed by the transformed host cell; (d) treating the stearoyl desaturase with a compound to be tested; and (e) comparing the activity of the stearoyl desaturase that has been treated with a test compound to the activity of an untreated stearoyl desaturase, thereby selecting compounds with potential for inhibitory activity.